The current project will investigate the regulation of an innate immunological protein, CD14, by vitamin D in mammary epithelial cells. Although actions of vitamin D in immune cells are well recognized, effects of vitamin D on the immune responses of epithelial cells have not been well studied. Preliminary data with human mammary epithelial (hTERT-HME) cells demonstrated that vitamin D induces expression and secretion of CD14, a protein that bind bacterial lipopolysaccharide (LPS). The proposed study will clarify the effect of vitamin D on CD14 and the innate immune response in mammary epithelial cells. The central hypothesis is that vitamin D promotes an anti-inflammatory environment through VDR to stimulate of soluble CD14 release from mammary epithelial cells. In Aim 1 we will determine if VDR is required for the induction of CD14 synthesis in response to physiological doses of vitamin D and, if so, the relative contributions of VDR signaling in the nucleus and at the plasma membrane. For this aim we will knock down VDR in the hTERT- HME cells with shRNA constructs and utilize vitamin D analogs that selectively activate nuclear or membrane VDRs activity. Outcomes will include CD14 gene expression by qPCR and sCD14 secretion by ELISA. In Aim 2 we will assess whether vitamin D alters the expression of membrane CD14 (mCD14) and characterize the isoforms of soluble CD14 (sCD14) secreted in response to vitamin D in hTERT-HME cells. These cells will be treated with concentrations of 1,25D (the VDR ligand) or 25D (the circulating vitamin D metabolite that will mimic physiological circulating concentrations in humans). Outcomes will include microscopy for mCD14 expression and western blotting to identify specific sCD14 isoforms. Because mCD14 is tethered to the membrane by a glycosylphosphatidylinositol (GPI) anchor, we will examine whether genomically inhibiting GPI synthesis blocks the effects of vitamin D. We will also use a chemical inhibitor to block the secretory pathway to determine its impact on vitamin D-induced sCD14. In Aim 3 we will investigate the functionality of sCD14 released from mammary cells in response to vitamin D. We will test the effects of conditioned media derived from vitamin D-treated hTERT-HME cells on immune responses of THP-1 macrophages. The THP-1 cells will be incubated in the condition media and challenged with LPS. The expression and secretion of pro- and anti- inflammatory cytokines by THP-1 cells will be assessed by qPCR and ELISA. We will also determine if the vitamin D-induced sCD14 alters binding or release of LPS from the macrophage by flow cytometry. We anticipate that the proposed studies will demonstrate that vitamin D triggers mammary epithelial cells to secrete sCD14 and promotes an anti-inflammatory environment. We will define the dose response of 25D with respect to anti-inflammatory responses, which will provide insight into whether vitamin D deficiency or vitamin D excess might alter these responses in vivo. Overall this work will enhance our understanding of the immunological functions of vitamin D in epithelial tissues.